【作者】 史卫卫；

【导师】 何琪杨；

【作者基本信息】 中国协和医科大学 ， 微生物与生化药学， 2010， 博士

【摘要】 肿瘤化学治疗最大障碍之一是肿瘤细胞对药物的耐受性。为了减少肿瘤耐药性的产生、降低药物毒副作用、增强药物疗效,一般采用联合用药。联合用药是近年来治疗肿瘤的重要进展之一,已成为肿瘤治疗的趋势,并得到广泛的应用。白藜芦醇(Resveratrol, RES)是植物产生的一种抗毒素,是非黄酮类多酚天然化合物,具有抗炎症、抗氧化、抗肿瘤等生物活性。白藜芦醇在抗肿瘤及心血管疾病方面具有非常巨大的应用价值。吉西他滨(Gemcitabine, GEM)化学名称为双氟脱氧胞苷,是脱氧胞嘧啶类似物,在细胞内活化后参入DNA合成,同时抑制核苷酸还原酶和脱氧胞嘧啶脱氨酶活性,是细胞周期特异性抗代谢类药物。本研究研探索了白藜芦醇增强吉西他滨体内外抗肿瘤疗效,及其增效作用的分子机制,为提高吉西他滨的临床抗肿瘤效果,减少不良反应发生提供指导意义。同时,对白藜芦醇增强DNA损伤药物抗肿瘤活性,及几个白藜芦醇新衍生物的抗肿瘤效果进行了初探性研究。1.RES增强GEM对多种细胞系的体外生长抑制作用我们选用低剂量吉西他滨与白藜芦醇联合作用于11种细胞,包括人正常细胞系：人胚肺成纤维细胞系2BS,人肝细胞系L02；人肿瘤细胞系：人肝癌细胞系HepG2和Bel-7402,人非小细胞肺癌细胞系A549,人结肠癌细胞系HCT116(包括p53野生型wild type, wt和p53突变型mutant,ko),人喉癌系KB及其耐药株KBV,人子宫颈癌细胞系HeLa；小鼠肝癌细胞系H22。我们发现RES不会增强GEM对正常细胞(2BS、L02)的生长抑制作用,即药物合用降低了GEM对正常细胞的毒性；RES与GEM合用除了对HeLa、HCT116 wt细胞没有增效作用,对其他几株细胞均有不同程度的协同增效作用,尤其对HepG2细胞的增效作用最为显著。20μM RES与0.5μM GEM联合用药指数CI值为0.52,具有显著协同增效作用。接下来我们在小鼠体内检测了RES联合GEM对H22移植瘤的体内生长抑制增效作用。结果表明RES(10 mg／kg)与GEM(25 mg／kg)低剂量合用,两药合用CI为0.73；RES(10 mg／kg)与GEM(50 mg／kg)高剂量合用,两药合用CI为0.56,RES与GEM合用体内抑瘤协同作用非常显著。2.RES增强GEM诱导肝癌HepG2细胞凋亡作用我们使用染色质凝集、流式检测PI染色、western blot几种实验方法,证明了RES能增强GEM诱导HepG2细胞凋亡的作用。20μM RES使0.5μM GEM诱导的HepG2凋亡细胞比例显著升高。3.RES增强GEM诱导肝癌HepG2细胞凋亡作用机制我们的研究发现RES能增强GEM体内外抗肝癌作用。接着我们进一步研究了增效作用的机制。结果表明RES增强GEM对HepG2细胞抑制作用不依赖于SIRT1、p53；RES增强GEM对HepG2细胞抑制作用与RES增强GEM诱导的HepG2细胞S期周期阻滞,及细胞内ROS水平升高有关；RES能下调HepG2内NF-KB表达水平,上调NF-κB抑制因子IκBα蛋白水平；且RES与GEM合用后,能显著下调NF-κB下游促存活靶蛋白：Survivin、Bcl-2、Bcl-xL、c-myc、Cyclin D1表达水平,上调促凋亡蛋白因子Bax蛋白水平,表明RES通过调节NF-κB及其靶蛋白水平来增强GEM对HepG2细胞凋亡的诱导作用。4.较低剂量下合用,RES增强GEM诱导肝癌HepG2细胞衰老样表型(SLP)发生我们使用低剂量RES(10μM)与低剂量GEM(0.05μM、0.1μM)和用处理HepG2细胞发现,在选用合用剂量下,RES没有增强GEM诱导的HepG2细胞凋亡,而是促使HepG2细胞衰老,生长停滞。因此低剂量下和用,RES同样增强了GEM对HepG2细胞的生长抑制作用。5.RES增强DNA损伤药物抗肿瘤活性接着我们又探索了RES与DNA损伤药物：博莱霉素(Bleomycin, BLM)、博安霉素(Boanmycin, BAM)、NC0604、多柔比星(DOX)联合用药对几株细胞的生长抑制作用。结果表明RES与博莱霉素家族抗生素联合作用于HepG2细胞,在一定浓度范围内具有协同增效作用,但增效作用并不显著；与DNA断裂药物DOX联合作用于HepG2细胞具有显著增效作用；RES与BAM、NC0604、DOX联用于Bel-7402细胞,也具有一定的协同增效作用,而RES与BAM联合用作用于HeLa细胞,CI值均大于1,表明两药合用拮抗,这与RES、GEM联合处理HeLa细胞结果相似,表明HeLa细胞对RES与GEM、BAM合用并不敏感,反而耐受。RES与BAM联合用药对小鼠体内H22移植瘤的抑制研究结果表明,RES(10mg／kg)、BAM(10 mg／kg)联合用药组与BAM(10 mg／kg)单药组相比较,小鼠瘤重有显著差异(P<0.05),联合用药指数CI值为0.73,具有协同增效作用。同时我们对NC0604抗肿瘤活性机制进行了初步探索,发现NC0604能损伤HepG2细胞DNA,通过影响调控细胞凋亡、存活的蛋白因子表达水平,来诱导细胞发生凋亡6.三种白藜芦醇衍生物抗肿瘤活性及联合用药初探白藜芦醇衍生物B1、B4、B5对HepG2细胞具有不同程度的生长抑制作用。其中B1、B4,尤其B4,抑瘤效果显著强于RES,具有进一步研究、开发的价值。接着我们将抑瘤效果相对较差的B1、B5与GEM、DNA损伤药物(BAM、NC0604、DOX)联合处理HepG2细胞,结果表明B1、B5与所选药物联合用药作用于HepG2细胞,合用效果均不理想。以上研究表明,白藜芦醇作为抗肿瘤化学增敏剂,具有潜在的临床应用价值。更多还原

【Abstract】 Despite aggressive therapies, resistance of many tumors to current treatment protocols still constitutes a major problem in cancer chemotherapy. To potentiate the efficacy and decrease the resistance and side effect of antitumor drugs, drug combination is widely used clinically. Drug combination is an important progress in the treatment of cancer in recent years and has become the trend of cancer treatment.Resveratrol (RES), a plant produced anti-toxin, is a kind of non-flavonoid polyphenol, natural compound with anti-inflammatory, antioxidant, anti-tumor and some other bio-logical activity. Resveratrol has a very great value in anti-tumor and cardiovascular dis-ease.Gemcitabine (GEM) is a deoxycytidine analogue. It participates in DNA synthesis af-ter activation in the cell. Meanwhile, it inhibits the activity of ribonucleotide reductase and deoxycytidine deaminase. And it is a cell cycle specific anti-metabolic drug.In this study, we explored the effect of resveratrol on potentiating anti-tumor activity of gemcitabine in vivo and in vitro, and the molecular mechanism of the synergy with Gemcitabine. This research provides guidance for enhancing the anti-tumor effect and reducing the incidence of adverse reaction of gemcitabine clinically. Meanwhile, we studied the activity of resveratrol enhancing the anti-tumor effect of DNA damage drugs, and the anti-tumor effect of several new derivatives of resveratrol.1. The effect of RES on enhancing anti-tumor activity of GEM in vivo and in vitroWe explored the synergy of RES and GEM on 11 kinds of cell lines, including human normal cell lines, such as embryonic lung fibroblast cell line 2BS, human liver cell line L02, and human tumor cell lines, such as human hepatoma cell line HepG2 and Bel-7402, human non-small cell lung cancer cell line A549, human colon cancer cell line HCT116 (p53 wide type:wt, and p53 mutant:ko), human laryngeal carcinoma line KB and its drug resistant strain KBV, human cervical cancer cell line HeLa, mouse hepatoma cell line H22. We found that RES did not enhance the effect of GEM on inhibition in human normal cell lines, which means drug combination reduced the cytotoxicity of GEM to normal cells. Except for HeLa and HCT116 wt, RES combination with GEM had differ-ent levels of synergies on the other several tumor cell lines, in particular, the synergy of HepG2 cells was most significant with CI value of 0.52.Then we examined the synergic effect of RES combination with GEM on H22 tumor growth inhibition in mice. The results showed that RES(10mg/kg) combination with GEM at low dose (25mg/kg) and high dose (50mg/kg) had the CI value of 0.73 and 0.56 respectively, which means RES combination with GEM synergy is very significant in vivi.2. RES potentiates the effect of GEM on apoptosis induction in HepG2 cells.The results of heochst33258 stain、PI stain、western blot, showed RES enhanced the apoptosis induction activity of GEM in HepG2 cells.3. The molecular mechanism of the synergy of RES combination with GEMWe further studied the mechanism of synergy. The results showed that RES potentiate the inhibition effect of GEM in HepG2 cells in SIRT1-and p53-independent way, but it is associated with cell cycle S phase arrest and elevated levels of intracellular ROS; RES can reduce the expression of NF-κB and increase IκBαlevel in HepG2 cells; RES combination with GEM, reduced the downstream target proteins of NF-κB, including Survivin、Bcl-2、Bcl-xL、c-myc、Cyclin D1, while Bax level was promoted. The data indicated that RES enhance apoptosis induction activity of GEM in HepG2 cells and is associated with the inhibition of NF-κB signal pathway.4. Low dose combination of RES and GEM induces senescence-like phenotype occurred in HepG2 cells.We treated HepG2 cells with low dose of RES(10μM) and low doses of GEM(0.05、0.1μM) and found RES did not enhance the GEM-induced apoptosis in HepG2 cells, but led HepG2 cells senescence and growth arrest. Therefore, low dose RES also enhanced the inhibition effect of GEM on HepG2 cells growth.5. RES enhances the anti-tumor activity of DNA damage drugsWe also explored the synergy of RES combination with DNA damage drugs, including Bleomycin(BLM)、Boanmycin(BAM)、NC0604、Doxorubicin(DOX), in several tumor cell lines. The result showed RES can enhance anti-tumor activity of bleomycin family in a certain concentration range, but not significantly; however the synergy of RES and DOX combination was significant; RES combination with BAM、NC0604、DOX was synergic at a certain level in Bel-7402 cells, but antagonistic in HeLa cells with CI value greater than 1.RES (10mg/kg) combination with BAM (10mg/kg) on mice H22 tumor inhibition re-sults showed that, they were synergic with CI value of 0.73.At the same time we explored the anti-tumor activity of NC0604. The results showed DNA was damaged by NC0604 in HepG2 cells, and apoptosis was induced by NC0604 in HepG2 cells through regulating apoptosis associated proteins.6. The preliminary exploration of three resveratrol derivatives and the combination of anti-tumor.Resveratrol derivatives B1, B4, B5 showed different levels of growth inhibition in HepG2 cells. The anti-tumor effects of B1, B4 were stronger than RES significantly, de-serving further study. However, the results of B1、B5 combination with DNA damage drugs(BAM、NC0604、DOX) in HepG2 cells, were not satisfactory.This study showed that resveratrol as an anti-tumor sensitizer, has a great potential value in clinical therapy.更多还原